Compositions And Methods For Identifying Modulars Of TRPV2

ABSTRACT

It has now been discovered that certain cannabinoids specifically activate TRPV2 channel activity. Based on the discovery, novel compositions and methods for screening, identifying and characterizing compounds that increase or decrease the biological activity of a TRPV2.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Application No. 60/731,686 filed onOct. 31, 2005 and Application No. 60/782,656 filed on Mar. 15, 2006, theentire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the regulation of thermal receptor ionchannel proteins. In particular, the present invention relates tocompositions and methods for screening, identifying and characterizingcompounds that increase or decrease the biological activity of a TRPV2.

BACKGROUND

In mammals, the sensation of pain triggered by thermal, mechanical orchemical stimuli is a useful warning and protective system. Considerableefforts have been put into elucidating the biochemical mechanismsinvolved in the detection, transduction and transmission of hot and coldsensations in neuronal tissues. Thermal stimuli activate specializedreceptors located on sensory neurons, such as those deriving from thedorsal root ganglion (DRG) and the trigeminal ganglion (TG). When thesestimuli are in the noxious range (i.e., very hot or cold), they activatea certain subset of thermal receptors on a sub-population of sensoryneurons called nociceptors (pain-sensing neurons). Upon activation, thethermal receptors (e.g., ion channels) transduce the noxious stimulusinto an electrical signal that is propagated along the sensory neuron tothe spinal cord, where it is relayed to the brain, ultimately leading tothe perception of pain. Accordingly, these thermal receptors representhighly promising targets for developing drugs for the treatment ofvarious painful conditions.

Several temperature-activated receptors have been identified with wideranging temperature sensitivities from noxious heat to noxious cold.These temperature-activated receptors belong to the transient receptorpotential (TRP) family of non-selective cation channels, which in C.elegans and D. melanogaster are involved in mechano- and osmoregulation.Several of these temperature-activated receptors, including TRPV1 andTRPV2, are implicated in noxious heat sensation (Caterina et al., 1997,Nature, 389: 816; and Caterina et al., 1999, Nature 398: 436). TRPV1,the most extensively characterized member of the thermo-TRP family, isactivated by moderate heat (˜43° C.), capsaicin, protons and certainendocannabinoids, such as anandamide and 2-AG. It is well accepted thatTRPV1 contributes to acute thermal nociception and hyperalgesia afterinjury (Clapham, Nature. 2003, 426(6966): 517-24).

TRPV2, also termed VRL-1, has been proposed as a sensor of noxioustemperatures (>52° C.), which presumably mediates “first” pain, i.e. therapid, acute, and sharp pain evoked by noxious stimuli (Caterina et al.,1999, supra; Story et al., Cell, 2003, 112:819-829, and referencestherein). TRPV2 is structurally most closely related to TRPV1 (˜50%sequence identity at the protein level). TRPV2 is expressed in medium-to large-diameter neurons of sensory ganglia, as well as at lower levelsin brain, spinal cord, spleen and lung. Furthermore, TRPV2 isupregulated in sympathetic postganglionic neurons following injury,suggesting a potential role for TRPV2 in sympathetically mediated pain(Gaudet et al., Brain Res. 2004, 1017(1-2):155-62). Thus, modulation ofTRPV2 may potentially have many therapeutic applications.

Despite great interest in TRPV2 modulation, a system for screening,identifying and characterizing TRPV2 modulators has yet to be developed.This is in part due to the lack of known, and in particular, selectiveTRPV2 agonists, as well as the technical difficulty of assaying thesechannels in a high temperature environment. In general, TRPV2 does notrespond to known TRPV1 agonists (Benham et al., 2003, Cell Calcium33:479-487). However, a recent study reported that 2-aminoethoxydiphenylborate (2-APB), a non-selective TRP modulator, was able to activateTRPV1, TRPV2, and TRPV3 (Hu et al (2004), J. Biol. Chem., 279: 35741-8),although TRPV2 activation by 2-APB was not observed by others (Chung etal. (2004), J Neurosci. 24: 5177-82).

In an effort to overcome the above-mentioned challenges, the presentinvention provides novel compositions and methods for screening,identifying and characterizing TRPV2 agonists.

SUMMARY

It has now been discovered that certain cannabinoids specificallyactivate TRPV2 channel activity.

In one general aspect, the present invention provides a method foridentifying a compound that decreases the biological activity of TRPV2,comprising the steps of: a) contacting a TRPV2 polypeptide with acannabinoid that is capable of activating TRPV2 activity under acondition in which the TRPV2 is activated by the cannabinoid; b)contacting the TRPV2 polypeptide with a test compound; c) measuring thebiological activity of the TRPV2 in the presence of both the cannabinoidand the test compound; d) repeating step a); e) measuring the biologicalactivity of the TRPV2 in the presence of the cannabinoid but not thetest compound; and f) comparing the TRPV2 activity measured from step c)with that from step e); thereby identifying the compound that decreasesthe biological activity of TRPV2 when the TRPV2 activity measured fromstep c) is less than that from step e).

In another general aspect, the present invention provides a method foridentifying a compound that increases the biological activity of TRPV2,comprising the steps of: a) obtaining atomic coordinates defining athree-dimensional structure of a complex comprising a TRPV2 interactingwith a cannabinoid that is capable of activating the TRPV2; b)elucidating a structural relationship between the TRPV2 and theinteracting cannabinoid; c) designing a structural analog of thecannabinoid based on the structural relationship; d) synthesizing thestructural analog; and e) determining the extent to which the structuralanalog alters the biological activity of the TRPV2, thereby identifyingthe compound that increases the biological activity of TRPV2.

Another general aspect of the present invention is a method forincreasing the biological activity of a TRPV2, comprising the step ofcontacting the TRPV2 with a cannabinoid that is capable of activatingthe TRPV2 activity.

The present invention further provides a method for stimulating noxiousthermo-sensation in a subject, comprising administering to the subject apharmaceutical composition comprising an effective amount of acannabinoid that is capable of activating the TRPV2 activity, therebystimulating the noxious thermo-sensation in the subject.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the subclasses of cannabinoids present in Cannabis (Thakuret al., Life Sci. 2005 Oct. 17, Epub ahead of print).

FIG. 2 shows non-enzymatic formation of Δ⁹-THC from its precursor(Thakur et al., supra).

FIG. 3 shows the structures of two representative endocannabinoid(Thakur et al., supra).

FIG. 4 shows concentration-dependent activation of rat TRPV2 by Δ⁹-THCin a FLIPR assay.

FIG. 5 illustrates activation of both rat and human TRPV2 by Δ⁹-THC andsubsequent block of the Δ⁹-THC-activated currents by ruthenium red fromwhole-cell patch clamp studies.

FIG. 6 shows Δ⁹-THC activated deletion mutants of TRPV2 recombinantlyexpressed from HEK293 cells: (A) the N-terminal deletion mutants; and(B) the C-terminal deletion mutants.

FIG. 7 illustrates the activation of the human and rat TRPV2 chimerarecombinantly expressed from HEK293 cells: (A) the chimera was expressedindividually from the cells; and (B) the complementary effect of RRH andHRR when they were co-expressed from the cells.

DETAILED DESCRIPTION

All publications cited herein are hereby incorporated by reference.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains.

As used herein, the terms “comprising”, “containing”, “having” and“including” are used in their open, non-limiting sense.

The following are abbreviations that are at times used in thisspecification:

2-AG=2-arachidonylglycerol

AEA=anandamide=N-arachidonoylethanolamine

bp=base pair

cDNA=complementary DNA

Ca²⁺=calcium

Δ⁹-THC=Delta-9-tetrahydrocannabinol

DRG=dorsal root ganglion

FLIPR=fluorescence imaging plate reader

kb=kilobase; 1000 base pairs

PAGE=polyacrylamide gel electrophoresis

PCR=polymerase chain reaction

SDS=sodium dodecyl sulfate

TG=trigeminal ganglion

TRPV2=transient receptor potential cation channel, subfamily V, member 2

As used herein, the term “biological activity of a TRPV2” refers to anactivity exerted by the TRPV2 protein as determined in vivo or in vitro,according to standard techniques. Such an activity can be a directactivity such as the ability of a TRPV2 to bind to a ligand, such as acannabinoid or an analog thereof. The activity can be the conductivityof an ion channel formed by the TRPV2. The activity can also befunctional changes of cell physiology, such as calcium mobilization ornociceptive response of the cell. The biological activity of a TRPV2 canbe an indirect activity, such as a signal transduction activity mediatedby TRPV2 via its interaction with one or more than one additionalprotein or other molecule(s).

“Binding affinity” refers to the ability of two or more molecularentities to bind or interact with each other. The binding can be fromthe formation of one or more chemical bonds that results in continualand stable proximity of the two interacting entities. The binding canalso be based solely on physical affinities, which can be equallyeffective in co-localizing the two interacting entities. Examples ofphysical affinities and chemical bonds include but are not limited to,forces caused by electrical charge differences, hydrophobicity, hydrogenbonds, van der Waals force, ionic force, covalent linkages, andcombinations thereof. The state of proximity between the interactingentities can be transient or permanent, reversible or irreversible. Inany event, it is in contrast to and distinguishable from contact causedby natural random movement of two entities.

“Cannabinoid” includes any of various compounds that activate acannabinoid receptor or a structural analog of the compounds.

In one embodiment, “cannabinoid” includes herbal cannabinoids, a classof compounds that were originally extracted from the plant Cannabissativa L or a metabolite thereof. Cannabis sativa L. is one of theoldest known medicinal plants and has been extensively studied withrespect to its phytochemistry. The plant biosynthesizes a total of 483identified chemical entities belonging to different chemical classes(ElSohly, 2002, In: F. Grotenhermen and E. Russo, Editors, Cannabis andCannabinoids, Haworth Press, Binghamton (2002), pp. 27-36.), of whichthe cannabinoids are the most distinctive class of compounds, known toexist only in this plant. There are 66 known plant-derived cannabinoids(Thakur et al., Life Sci. 2005 Oct. 17, Epub ahead of print). The mostprevalent of which are the tetrahydrocannabinols (THCs), thecannabidiols (CBDs), and the cannabinols (CBNs). The next most abundantcannabinoids are the cannabigerols (CBGs), the cannabichromenes (CBCs),and cannabinodiols (CBNDs).

FIG. 1 shows the representative structures of subclasses of cannabinoidspresent in Cannabis sativa. Most cannabinoids contain 21 carbon atoms,but there are some variations in the length of the C-3 side chainattached to the aromatic ring. In the most common homologues, then-pentyl side chain is replaced with an n-propyl (De Zeeuw et al.,Science. 1972, 175:778-779); and Vree et al., Journal of Pharmacy andPharmacology. 1972, 24:7-12). These analogues are named using the suffix“varin” and are designated as THCV, CBDV, or CBNV, as examples.Cannabinoids with one (Vree et al., 1972, supra) and four (Smith, 1997,Journal of Forensic Sciences 42 (1997), pp. 610-618) carbons also existbut are minor components. Classical cannabinoids (CCs) are ABC tricyclicterpenoid compounds bearing a benzopyran moiety and are insoluble inwater but soluble in lipids, alcohols, and other non-polar organicsolvents (Thakur et al., 2005, supra). These phenolic derivatives aremore water-soluble as their phenolate salts formed under strong alkalineconditions.

One particular example of “cannabinoid” is Delta-9-tetrahydrocannabinol(Delta-9-THC, Δ⁹-THC), the key psychoactive ingredient of cannabis(marijuana) (Gaoni and Mechoulam 1964, Journal of the American ChemicalSociety 86 (1964), pp. 1646-1647). As illustrated in FIG. 2, Δ⁹-THC isformed by the decarboxylation of its non-psychoactive precursor Δ⁹-THCAby the action of light or heat during storage or smoking or underalkaline conditions. Δ⁹-THCA is biosynthesized by a well-establishedpathway involving the action of several specific enzymes.

It was discovered that Δ⁹-THC interacts with the two known cannabinoid(CB) receptors, CB1 (Devane et al., 1988, Molecular Pharmacology 34(1988), pp. 605-613; Gerard et al., 1990, Nucleic Acids Research 18(1990), p. 7142; Gerard et al., 1991, Biochemical Journal 279 (1991),pp. 129-134; and Matsuda et al., 1990, Nature 346 (1990), pp. 561-564.)and CB2 (Munro et al., 1993, Nature 365 (1993), pp. 61-65). Bothcannabinoid receptors belong to the super family of G-protein coupledreceptors, and produce a broad spectrum of physiological effects(Grotenhermen, 2002, In: R. Grotenhermen and E. Russo, Editors, Cannabisand Cannabinoids, Haworth Press, Binghamton (2002), pp. 123-142)including antiemetic, appetite enhancing, analgesic, and lowering ofintraocular pressure. The discovery of specific cannabinoid receptorsinside animal ultimately led to the search and identification ofendocannabinoid.

Thus, the term “cannabinoid” also includes endocannabinoid. The term“endocannabinoid” refers to a ligand to a cannabinoid receptor, whereinsaid ligand is endogenously produced by in the bodies of an animal.Exemplary endocannabinoids include, but are not limited to,N-arachidonoylethanolamine (AEA, anandamide) and 2-arachidonylglycerol(2-AG), the structures of which are shown in FIG. 3. Anandamide wasshown to bind to the CB1 receptor with modest affinity (K_(i)=61 nM),have low affinity for the CB2 receptor (K_(i)=1930 nM) (Lin et al.,1998, Journal of Medicinal Chemistry 41 (1998), pp. 5353-5361), andbehave as a partial agonist in the biochemical and pharmacological testsused to characterize cannabinoid activity. It was reported thatanandamide can also bind to and activate TRPV1 (Di Marzo et al.,Prostaglandins Leukot Essent Fatty Acids 2002; 66: 377-91). 2-AG bindsweakly to both CB1 (K_(i)=472 nM) and CB2 (K_(i)=1400 nM) receptors(Mechoulam et al., 1995, Biochemical Pharmacology 50 (1995), pp. 83-90).2-AG was isolated from intestinal and brain tissues and is present inthe brain at concentrations approximately 170-fold higher than AEA (3)(Stella et al., 1997, Nature 388 (1997), pp. 773-778).

In yet another embodiment, the term “cannabinoid” covers the syntheticcannabinoids are produced by chemical synthesis and do not occurnaturally. The synthetic cannabinoids can be synthesized based on thestructure of herbal cannabinoids or endocannabinoid. Syntheticcannabinoids are particularly useful in experiments to determine therelationship between the structure and activity of cannabinoidcompounds, by making systematic, incremental modifications ofcannabinoid molecule. Exemplary synthetic cannabinoids includesdronabinol (synthetic THC), nabilone, and any other synthetic compoundsthat activate a cannabinoid receptor or a structural analog of thecompounds.

A “cannabinoid that is capable of activating the TRPV2 activity” refersto any cannabinoid that is capable of binding to a TRPV2 channel and, inthe absence of other stimulation, exhibits at least a 10% increase inthe conductivity of the TRPV2 channel compared to the baseline. A personskilled in the art can experimentally determine whether a cannabinoid iscapable of activating the TRPV2 activity. In some embodiments,“cannabinoid that is capable of activating the TRPV2 activity” is acannabinoid which, upon binding to a TRPV2 channel, results in at leasta 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in the conductivityof the channel compared to the baseline. “Cannabinoid that is capable ofactivating the TRPV2 activity” includes, but is not limited to,Δ⁹-tetrahydrocannabinol, cannabinol, cannabidiol nabilone, CP55940,HU210, and 2-AG. Interestingly, the other endocannabinoid tested,anandamide, showed no or minimal activation effect on TRPV2 (Table 2,Example 4 infra).

A “cannabinoid receptor” or a “CB receptor” each refers to a proteinthat functions as a specific receptor for a cannabinoid. The “CBreceptor” can be a CB1 receptor or a CB2 receptor.

The CB1 receptor has been detected primarily in brain, specifically inthe basal ganglia and in the limbic system, including the hippocampus.They are also found in other tissues such as the cerebellum and in bothmale and female reproductive systems. CB1 receptors appear to beresponsible for the euphoric and anticonvulsive effects of cannabis. ACB1 can (1) have greater than about 70% amino acid sequence identity toa human CB1 receptor depicted in GenBank protein ID: NP_(—)057167 (thelonger isoform of human CB1 receptor) or NP_(—)149421 (the shorterisoform of human CB1 receptor); or (2) bind to antibodies, e.g.,polyclonal or monoclonal antibodies, raised against the human CB1receptor depicted in GenBank protein ID NP_(—)057167 or NP_(—)149421. Insome embodiments, the CB1 receptor has greater than about 75, 80, 85,90, or 95 percent amino acid sequence identity to the human CB1 receptordepicted in GenBank protein ID NP_(—)057167 or NP_(—)149421. The CB1receptor includes orthologs of the CB1 receptors in animals includinghuman, rat, mouse, pig, dog and monkey, etc. The CB1 receptor alsoincludes structural and functional polymorphisms of the CB1 receptor.“Polymorphism” refers to a set of genetic variants at a particulargenetic locus among individuals in a population. The CB1 receptorincludes the structural and functional polymorphisms of the CB1 receptorfrom human (GenBank protein ID NP_(—)057167 or NP_(—)149421), rat(GenBank protein ID: NP_(—)036916), or mouse (GenBank protein ID:NP_(—)031752), or etc.

The CB2 receptor has been detected almost exclusively in the immunesystem, with the greatest density in the peripheral blood cells. CB2receptors appear to be responsible for the anti-inflammatory andpossible other therapeutic. A CB2 can (1) have greater than about 70%amino acid sequence identity to a human CB2 receptor depicted in GenBankprotein ID: NP_(—)001832; or (2) bind to antibodies, e.g., polyclonal ormonoclonal antibodies, raised against the human CB2 receptor depicted inGenBank protein ID NP_(—)001832. In some embodiments, the CB2 receptorhas greater than about 75, 80, 85, 90, or 95 percent amino acid sequenceidentity to the human CB2 receptor depicted in GenBank protein IDNP_(—)001832. The CB2 receptor includes orthologs of the CB2 receptorsin animals including human, rat, mouse, pig, dog and monkey, etc. TheCB2 receptor also includes structural and functional polymorphisms ofthe CB2 receptor. The CB2 receptor includes the structural andfunctional polymorphisms of the CB2 receptor from human (GenBank proteinID NP_(—)001832), rat (GenBank protein ID: NP_(—)065418), mouse (GenBankprotein ID: NP_(—)034054), or etc.

A “cell” refers to at least one cell or a plurality of cells appropriatefor the sensitivity of the detection method. The cell can be present ina cultivated cell culture. The cell can also be present in its naturalenvironment, such as a biological tissue or fluid. Cells suitable forthe present invention may be bacterial, but are preferably eukaryotic,and are most preferably mammalian.

A “compound that increases the conductivity of a TRPV2 channel” includesany compound that results in increased passage of ions through the TRPV2channel. In one embodiment, such a compound is an agonist for the TRPV2channel that binds to the TRPV2 channel to increase its conductivity.Such a compound triggers, initiates, propagates, or otherwise enhancesthe channel conductivity. In another embodiment, such a compound is apositive allosteric modulator, which interacts with the TRPV2 channel atallosteric sites different from the agonist-binding site, andpotentiates the response of the channel to an agonist.

A “compound that decreases the conductivity of a TRPV2 channel” includesany compound that results in decreased passage of ions through the TRPV2channel. In one embodiment, such a compound is an antagonist for theTRPV2 channel that binds to the TRPV2 channel to counter, decrease orlimit the action of an agonist in either a competitive ornon-competitive fashion. In another embodiment, such a compound is anegative allosteric modulator, which interacts with the TRPV2 channel atallosteric sites different from the agonist or antagonist-binding site,and decreases the response of the channel to an agonist. In yet anotherembodiment, such a compound is an inverse agonist that binds to theTRPV2 channel and decreases the conductivity of the channel in theabsence of any other compound, such as an agonist.

“Nucleotide sequence” refers to the arrangement of eitherdeoxyribonucleotide or ribonucleotide residues in a polymer in eithersingle- or double-stranded form. Nucleic acid sequences can be composedof natural nucleotides of the following bases: thymidine, adenine,cytosine, guanine, and uracil; abbreviated T, A, C, G, and U,respectively, and/or synthetic analogs of the natural nucleotides.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from at least one of the other nucleic acid molecules presentin the natural source of the nucleic acid, or is substantially free ofat least one of the chemical precursors or other chemicals when thenucleic acid molecule is chemically synthesized. An “isolated” nucleicacid molecule can also be, for example, a nucleic acid molecule that issubstantially free of at least one of the nucleotide sequences thatnaturally flank the nucleic acid molecule at its 5′ and 3′ ends in thegenomic DNA of the organism from which the nucleic acid is derived. Anucleic acid molecule is “substantially separated from” or“substantially free of” other nucleic acid molecule(s) or otherchemical(s) in preparations of the nucleic acid molecule when there isless than about 30%, 20%, 10%, or 5% (by dry weight) of the othernucleic acid molecule(s) or the other chemical(s) (also referred toherein as a “contaminating nucleic acid molecule” or a “contaminatingchemical”).

Isolated nucleic acid molecules include, without limitation, separatenucleic acid molecules (e.g., cDNA or genomic DNA fragments produced byPCR or restriction endonuclease treatment) independent of othersequences, as well as nucleic acid molecules that are incorporated intoa vector, an autonomously replicating plasmid, a virus (e.g., aretrovirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid moleculecan include a nucleic acid molecule that is part of a hybrid or fusionnucleic acid molecule. An isolated nucleic acid molecule can be anucleic acid sequence that is: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) synthesized by, for example,chemical synthesis; (iii) recombinantly produced by cloning; or (iv)purified, as by cleavage and electrophoretic or chromatographicseparation.

The term “oligonucleotide” or “oligo” refers to a single-stranded DNA orRNA sequence of a relatively short length, for example, less than 100residues long. For many methods, oligonucleotides of about 16-25nucleotides in length are useful, although longer oligonucleotides ofgreater than about 25 nucleotides may sometimes be utilized. Someoligonucleotides can be used as “primers” for the synthesis ofcomplimentary nucleic acid strands. For example, DNA primers canhybridize to a complimentary nucleic acid sequence to prime thesynthesis of a complimentary DNA strand in reactions using DNApolymerases. Oligonucleotides are also useful for hybridization inseveral methods of nucleic acid detection, for example, in Northernblotting or in situ hybridization.

The terms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinatedforms, etc. Modifications also include intra-molecular crosslinking andcovalent attachment to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by the codons of genes may also be included in a polypeptide.

An “isolated protein” is one that is substantially separated from atleast one of the other proteins present in the natural source of theprotein, or is substantially free of at least one of the chemicalprecursors or other chemicals when the protein is chemicallysynthesized. A protein is “substantially separated from” or“substantially free of” other protein(s) or other chemical(s) inpreparations of the protein when there is less than about 30%, 20%, 10%,or 5% (by dry weight) of the other protein(s) or the other chemical(s)(also referred to herein as a “contaminating protein” or a“contaminating chemical”).

Isolated proteins can have several different physical forms. Theisolated protein can exist as a full-length nascent or unprocessedpolypeptide, or as a partially processed polypeptide or as a combinationof processed polypeptides. The full-length nascent polypeptide can bepostranslationally modified by specific proteolytic cleavage events thatresult in the formation of fragments of the full-length nascentpolypeptide. A fragment, or physical association of fragments can havethe biological activity associated with the full-length polypeptide;however, the degree of biological activity associated with individualfragments can vary.

An isolated polypeptide can be a non-naturally occurring polypeptide.For example, an “isolated polypeptide” can be a “hybrid polypeptide.” An“isolated polypeptide” can also be a polypeptide derived from anaturally occurring polypeptide by additions or deletions orsubstitutions of amino acids. An isolated polypeptide can also be a“purified polypeptide” which is used herein to mean a specifiedpolypeptide in a substantially homogeneous preparation substantiallyfree of other cellular components, other polypeptides, viral materials,or culture medium, or when the polypeptide is chemically synthesized,chemical precursors or by-products associated with the chemicalsynthesis. A “purified polypeptide” can be obtained from natural orrecombinant host cells by standard purification techniques, or bychemical synthesis, as will be apparent to skilled artisans.

“Recombinant” refers to a nucleic acid, a protein encoded by a nucleicacid, a cell, or a viral particle, that has been modified usingmolecular biology techniques to something other than its natural state.For example, recombinant cells can contain nucleotide sequence that isnot found within the native (non-recombinant) form of the cell or canexpress native genes that are otherwise abnormally, under-expressed, ornot expressed at all. Recombinant cells can also contain genes found inthe native form of the cell wherein the genes are modified andre-introduced into the cell by artificial means. The term alsoencompasses cells that contain an endogenous nucleic acid that has beenmodified without removing the nucleic acid from the cell; suchmodifications include those obtained, for example, by gene replacement,and site-specific mutation.

A “recombinant host cell” is a cell that has had introduced into it arecombinant DNA sequence. Recombinant DNA sequence can be introducedinto host cells using any suitable method including, for example,electroporation, calcium phosphate precipitation, microinjection,transformation, biolistics and viral infection. Recombinant DNA may ormay not be integrated (covalently linked) into chromosomal DNA making upthe genome of the cell. For example, the recombinant DNA can bemaintained on an episomal element, such as a plasmid. Alternatively,with respect to a stably transformed or transfected cell, therecombinant DNA has become integrated into the chromosome so that it isinherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the stably transformed ortransfected cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA. Recombinanthost cells may be prokaryotic or eukaryotic, including bacteria such asE. coli, fungal cells such as yeast, mammalian cells such as cell linesof human, bovine, porcine, monkey and rodent origin, and insect cellssuch as Drosophila- and silkworm-derived cell lines. It is furtherunderstood that the term “recombinant host cell” refers not only to theparticular subject cell, but also to the progeny or potential progeny ofsuch a cell. Because certain modifications can occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

“Sequence identity or similarity”, as known in the art, is therelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Asused herein, “identity”, in the context of the relationship between twoor more nucleic acid sequences or two or more polypeptide sequences,refers to the percentage of nucleotide or amino acid residues,respectively, that are the same when the sequences are optimally alignedand analyzed. For purposes of comparing a queried sequence against, forexample, the amino acid sequence SEQ ID NO:2, the queried sequence isoptimally aligned with SEQ ID NO: 2 and the best local alignment overthe entire length of SEQ ID NO:2 is obtained.

Analysis can be carried out manually or using sequence comparisonalgorithms. For sequence comparison, typically one sequence acts as areference sequence, to which a queried sequence is compared. When usinga sequence comparison algorithm, test and reference sequences are inputinto a computer, sub-sequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated.

Optimal alignment of sequences for comparison can be conducted, forexample, by using the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol., 48:443 (1970). Software for performing Needleman& Wunsch analyses is publicly available through the Institut Pasteur(France) Biological Software website:http://bioweb.pasteur.fr/seqanal/interfaces/needle.html. The NEEDLEprogram uses the Needleman-Wunsch global alignment algorithm to find theoptimum alignment (including gaps) of two sequences when consideringtheir entire length. The identity is calculated along with thepercentage of identical matches between the two sequences over thereported aligned region, including any gaps in the length. Similarityscores are also provided wherein the similarity is calculated as thepercentage of matches between the two sequences over the reportedaligned region, including any gaps in the length. Standard comparisonsutilize the EBLOSUM62 matrix for protein sequences and the EDNAFULLmatrix for nucleotide sequences. The gap open penalty is the score takenaway when a gap is created; the default setting using the gap openpenalty is 10.0. For gap extension, a penalty is added to the standardgap penalty for each base or residue in the gap; the default setting is0.5.

Hybridization can also be used as a test to indicate that twopolynucleotides are substantially identical to each other.Polynucleotides that share a high degree of identity will hybridize toeach other under stringent hybridization conditions. “Stringenthybridization conditions” has the meaning known in the art, as describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1989). An exemplary stringent hybridization condition compriseshybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC and 0.1% SDS at 50-65° C.,depending upon the length over which the hybridizing polynucleotidesshare complementarity.

A “TRPV2”, “transient receptor potential cation channel, subfamily V,member 2”, “VRL”, “VRL1”, “VRL-1”, or “vanilloid receptor-like protein1” each refers to a protein that forms an ion channel, the TRPV2channel, that can be activated by high temperature and/or lowosmolarity, and transduces heat responses in sensory ganglia. The TRPV2channel can also be activated by certain compounds. An activated TRPV2channel gates the influx of Ca²⁺ and other cations (e.g., Na⁺) throughthe channel, resulting in membrane depolarization. A TRPV2 protein can(1) have greater than about 70% amino acid sequence identity to a humanTRPV2 (hTRPV2) protein depicted in SEQ ID NO: 2 (GenBank protein ID:NP_(—)057197); or (2) bind to antibodies, e.g., polyclonal or monoclonalantibodies, raised against a hTRPV2 protein depicted in SEQ ID NO: 2. Insome embodiments, the TRPV2 has greater than about 75, 80, 85, 90, or 95percent amino acid sequence identity to SEQ ID NO: 2. TRPV2 includesorthologs of the TRPV2 in animals including human, rat, mouse, pig, dogand monkey, etc. TRPV2 also includes structural and functionalpolymorphisms of the TRPV2. TRPV2 includes the structural and functionalpolymorphisms of the TRPV2 from human, rat (GenBank protein ID:NP_(—)058903, SEQ ID NO:4), mouse (GenBank protein ID: NP_(—)035836, SEQID NO:6), or etc. For example, it was found that addition of ahemagglutinin A (HA) epitope tag to the end of the rat TRPV2 C-terminusdid not alter the channel properties; and that deletion mutants of ratTRPV2-HA lacking the N-terminal 20, 32, and 65 and C-terminal 11, 23, or32 amino acid residues of rat TRPV2 were still active in their responsesto an elevated temperature of about 53° C., lowered osmolarity, Δ9-THCor 2-APB. Therefore, TRPV2 also includes deletion or modifications ofthe wild-type TRPV2 that maintains the biological activity of the TRPV2,such as the deletion mutants of rat TRPV2 consisting of the amino acidsequence of SEQ ID NOs: 7-14. Furthermore, TRPV2 also includes chimerasbetween TRPV2 of different animals. For example, it was found thatchimeras (SEQ ID NO: 16) between rat and human TRPV2, named RHR (i.e.Rat 1-392/Human 391-646/Rat 647-761), was also active in its response toan elevation of temperature of about 53° C., Δ⁹-THC and to 2-APB. Inaddition, TRPV2 further includes an active ion channel formed by thecombination of two or more TRPV2 subunits, which by themselves areinactive or less active. For example, TRPV2 can be an active ion channelformed by the co-expression of the chimera RRH (Rat 1-392/Rat393-646/Human 647-764) and HRR (Human 1-390/Rat 393-646/Rat 647-761).

“TRPV2 activation temperature” is the temperature at which a TRPV2channel, in the absence of other stimulation, exhibits at least a 10%increase in its conductivity compared to the baseline. A person skilledin the art can experimentally determine the activation temperature for aTRPV2 channel. In some embodiments, “TRPV2 activation temperature” isthe temperature at which a TRPV2 channel exhibits at least a 15%, 20%,25%, 30%, 35%, 40%, 45%, or 50% increase in its conductivity compared tothe baseline. “TRPV2 activation temperature” is typically greater thanof about 52° C. In some embodiments, the TRPV2 activation temperature isabout 52° C.-55° C. or 55° C.-60° C.

“TRPV2 non-activation temperature” is the temperature that falls outsideof the range for a “TRPV2 activation temperature”. An exemplary TRPV2non-activation temperature is room temperature (about 22° C.) or anytemperature that is below about 52° C.

“Vector” refers to a nucleic acid molecule into which a heterologousnucleic acid can be or is inserted. Some vectors can be introduced intoa host cell allowing for replication of the vector or for expression ofa protein that is encoded by the vector or construct. Vectors typicallyhave selectable markers, for example, genes that encode proteinsallowing for drug resistance, origins of replication sequences, andmultiple cloning sites that allow for insertion of a heterologoussequence. Vectors are typically plasmid-based and are designated by alower case “p” followed by a combination of letters and/or numbers.Starting plasmids disclosed herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids by application of procedures known in the art. Manyplasmids and other cloning and expression vectors that can be used inaccordance with the present invention are well-known and readilyavailable to those of skill in the art. Moreover, those of skill readilymay construct any number of other plasmids suitable for use in theinvention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

“Sequence” means the linear order in which monomers occur in a polymer,for example, the order of amino acids in a polypeptide or the order ofnucleotides in a polynucleotide.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology and recombinant DNA are used. Thesetechniques are well-known and are explained in, for example, CurrentProtocols in Molecular Biology, Vols. I, II, and III, F. M. Ausubel, ed.(1997); and Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

It was discovered in the present invention that a group of cannabinoidsare capable of activating the TRPV2 but not TRPV1 activity. Thus, thepresent invention provides new methods for regulating the biologicalactivity of TRPV2 and new methods for identifying compounds thatregulate biological activity of TRPV2.

In one embodiment, the TRPV2 used in the present invention is present ina cell. The cell can express TRPV2 endogeneously or recombinantly. Oneexemplary endogeneous cell for TRPV2 is a dorsal root ganglia (DRG)neuron or trigeminal neurons. Other examples of endogeneous cell forTRPV2 include, but are not limited to, intestine intrinsic neurons,vascular smooth muscle cells, and human hepatoblastoma (HepG2).

It will be apparent to skilled artisans that any recombinant expressionmethods may be used in the present invention for purposes of expressingthe TRPV2. Generally, a nucleic acid encoding TRPV2 can be introducedinto a suitable host cell. Exemplary nucleic acid molecules that can beused in the present invention include cDNA that encodes for the fulllength TRPV2 from human (SEQ ID: 1, GenBank accession No: NM_(—)016113),mouse (SEQ ID NO:5, GenBank accession No: NM_(—)011706), or rat (SEQ IDNO:3 GenBank accession No: NM_(—)017207).

Typically, the nucleic acids, preferably in the form of DNA, areincorporated into a vector to form expression vectors capable ofdirecting the production of the interacting protein member(s) onceintroduced into a host cell. Many types of vectors can be used for thepresent invention. Methods for the construction of an expression vectorfor purposes of this invention should be apparent to skilled artisansapprised of the present disclosure. (See generally, Current Protocols inMolecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRLPress, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods inEnzymology 153:516-544 (1987); The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II, 1982; and Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 1989.)

Generally, the expression vectors include an expression cassette havinga promoter operably linked to a DNA encoding an interacting proteinmember. The promoter can be a native promoter, i.e., the promoter foundin naturally occurring cells to be responsible for the expression of theinteracting protein member in the cells. Alternatively, the expressioncassette can be a chimeric one, i.e., having a heterologous promoterthat is not the native promoter responsible for the expression of theinteracting protein member in naturally occurring cells. The expressionvector may further include an origin of DNA replication for thereplication of the vectors in host cells. Preferably, the expressionvectors also include a replication origin for the amplification of thevectors in, e.g., E. coli, and selection marker(s) for selecting andmaintaining only those host cells harboring the expression vectors.

The thus constructed expression vectors can be introduced into the hostcells by any techniques known in the art, e.g., by direct DNAtransformation, microinjection, electroporation, viral infection,lipofection, gene gun, and the like. The expression of the protein ofinterest may be transient or stable. The expression vectors can bemaintained in host cells in an extrachromosomal state, i.e., asself-replicating plasmids or viruses. Alternatively, the expressionvectors can be integrated into chromosomes of the host cells byconventional techniques such as selection of stable cell lines orsite-specific recombination. In stable cell lines, at least theexpression cassette portion of the expression vector is integrated intoa chromosome of the host cells.

The vector construct can be designed to be suitable for expression invarious host cells, including but not limited to bacteria, yeast cells,plant cells, insect cells, and mammalian and human cells. Methods forpreparing expression vectors for expression in different host cellsshould be apparent to a skilled artisan. As described in the Example 1,infra, rat and human TRPV2 has been successfully expressed in HEK293.

Homologues and fragments of TRPV2 can also be easily expressed using therecombinant methods described above. For example, to express a proteinfragment, the DNA fragment incorporated into the expression vector canbe selected such that it only encodes the protein fragment. Likewise, aspecific hybrid protein can be expressed using a recombinant DNAencoding the hybrid protein. Similarly, a homologue protein may beexpressed from a DNA sequence encoding the homologue protein. Ahomologue-encoding DNA sequence may be obtained by manipulating thenative protein-encoding sequence using recombinant DNA techniques. Forthis purpose, random or site-directed mutagenesis can be conducted usingtechniques generally known in the art. To make protein derivatives, forexample, the amino acid sequence of a native interacting protein membermay be changed in predetermined manners by site-directed DNA mutagenesisto create or remove consensus sequences.

In other embodiments, the TRPV2 is provided in a cell membrane. Themembrane preparation can be isolated from a native host cell, forexample, a DRG or TG cell, which expresses TRPV2 on its cell surface.The membrane preparation can also be isolated from a recombinant hostcell, for example, a CHO, HEK293, or COS cell, which expresses a TRPV2recombinantly on its cell surface. The membrane preparation can befurther prepared from the biological membranes, such as the tissuemembrane, plasma membrane, cell membrane, or internal organelle membraneexpressing the TRPV2 channels. Methods are known to those skilled in theart for isolation and preparation of biological membrane preparations.For example, such a method can include the steps of mechanical orenzymatic disruption of the tissue or cells, centrifugation to separatethe membranes from other components, and resuspending the membranefragments or vesicles in suitable buffer solution. Alternatively, themembrane-containing preparation can also be derived from artificialmembranes. Purified TRPV2 protein can be reconstituted into lipidbilayers to form artificial membrane vesicles (see Chen et al., 1996, J.Gen. Physiol. 108:237-250). Such type of membrane vesicle can be veryspecific to the channel of interest, avoiding the problem ofcontamination from other channels. For example, such artificialmembranes can include an electrode to which is tethered a lipid membranecontaining ion channels and forming ion reservoirs. Methods are known tothose skilled in the art to prepare artificial membrane vesicles.

In some preferred embodiments, membranes can be broken under controlledconditions, yielding portions of cell membranes and/or membranevesicles. Cell membrane portions and/or vesicles can, in someembodiments, provide an easier format for the inventive assays andmethods, since cell lysis and/or shear is not as much of a concernduring the assay. Cell membranes can be derived from tissues and/orcultured cells. Such methods of breaking cell membranes and stabilizingthem are known in the art. Methods of treating tissues to obtain cellmembranes are known in the art.

Preferably, human TRPV2 is used in the assays of the invention.Optionally, TRPV2 orthologs from other species such as rat or mouse,preferably a mammalian species, are used in assays of the invention.

The compound identification methods can be performed using conventionallaboratory formats or in assays adapted for high throughput. The term“high throughput” refers to an assay design that allows easy screeningof multiple samples simultaneously and/or in rapid succession, and caninclude the capacity for robotic manipulation. Another desired featureof high throughput assays is an assay design that is optimized to reducereagent usage, or minimize the number of manipulations in order toachieve the analysis desired. Examples of assay formats include 96-wellor 384-well plates, levitating droplets, and “lab on a chip”microchannel chips used for liquid handling experiments. It is wellknown by those in the art that as miniaturization of plastic molds andliquid handling devices are advanced, or as improved assay devices aredesigned, greater numbers of samples can be processed using the designof the present invention.

Any test compounds may be screened in the screening assays of thepresent invention to select modulators of the protein complex of theinvention. By the term “selecting” or “select” compounds it is intendedto encompass both (a) choosing compounds from a group previously unknownto be modulators of a protein complex or interacting protein membersthereof, and (b) testing compounds that are known to be capable ofbinding, or modulating the functions and activities of, a proteincomplex or interacting protein members thereof. Both types of compoundsare generally referred to herein as “test compounds” or “candidatecompound”. The candidate compounds encompass numerous chemical classes,including but not limited to, small organic or inorganic compounds,natural or synthetic molecules, such as antibodies, proteins orfragments thereof, antisense nucleotides, interfering RNA (iRNA) andribozymes, and derivatives, mimetics and analogs thereof. Preferably,they are small organic compounds, i.e., those having a molecular weightof no greater than 10,000 daltons, more preferably less than 5,000daltons. Preferably, the test compounds are provided in library formatsknown in the art, e.g., in chemically synthesized libraries (Seegenerally, Gordan et al. J. Med. Chem., 37:1385-1401 (1994)),recombinantly expressed libraries (e.g., phage display libraries), andin vitro translation-based libraries (e.g., ribosome display libraries).

Candidate compounds comprise functional chemical groups necessary forstructural interactions with polypeptides, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, preferably atleast two of the functional chemical groups and more preferably at leastthree of the functional chemical groups. The candidate compounds cancomprise cyclic carbon or heterocyclic structure and/or aromatic orpolyaromatic structures substituted with one or more of theabove-identified functional groups. Candidate compounds also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the compound is anucleic acid, the compound typically is a DNA or RNA molecule, althoughmodified nucleic acids having non-natural bonds or subunits are alsocontemplated.

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random peptides, and the like. Candidatecompounds can also be obtained using any of the numerous approaches incombinatorial library methods known in the art, including biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries: synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection (Lam (1997) Anticancer Drug Des.12: 145). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural and synthetically produced libraries andcompounds can be readily modified through conventional chemical,physical, and biochemical means.

Further, known pharmacological agents can be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidation, etc. to produce structural analogs of theagents. Candidate compounds can be selected randomly or can be based onexisting compounds that bind to and/or modulate the function of TRPV2activity. Therefore, a source of candidate agents is one or more thanone library of molecules based on one or more than one known compoundthat increases or decreases TRPV2 channel conductivity in which thestructure of the compound is changed at one or more positions of themolecule to contain more or fewer chemical moieties or differentchemical moieties. The structural changes made to the molecules increating the libraries of analog activators/inhibitors can be directed,random, or a combination of both directed and random substitutionsand/or additions. One of ordinary skill in the art in the preparation ofcombinatorial libraries can readily prepare such libraries based on theexisting compounds.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. that can be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent canalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas nuclease inhibitors, antimicrobial agents, and the like can also beused.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: Zuckermann et al. (1994). J. Med.Chem. 37:2678. Libraries of compounds can be presented in solution(e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam(1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698),plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) orphage (see e.g., Scott and Smith (1990) Science 249:386-390).

The selected compounds can be tested for their ability to decrease thechannel conductivity of the TRPV2, or for their ability to decrease thebinding activity of the TRPV2 to a cannabinoid that is capable ofactivating the TRPV2. During the test, the test compound can be added tothe TRPV2 prior to, after, or simultaneously with cannabinoid that iscapable of activating the TRPV2. In addition, the compounds can betested in an animal model for pain, or inflammation, etc.

Generally, a control assay is performed in which the above screeningassay is conducted in the absence of the test compound. The result ofthis assay is then compared with that obtained in the presence of thetest compound.

The test compounds may be screened in an in vitro assay to identifycompounds capable of binding to a TRPV2. For this purpose, a testcompound is contacted with TRPV2 under conditions and for a timesufficient to allow specific interaction between the test compound andthe TRPV2 to occur, thereby resulting in the binding of the compound tothe TRPV2, and the formation of a complex. Subsequently, the bindingevent is detected.

In one particular embodiment, the TRPV2 is immobilized on a solidsupport (such as a protein microchip) or on a cell surface or amembrane. For example, the protein complex can be immobilized directlyonto a microchip substrate such as glass slides or onto multi-wellplates using non-neutralizing antibodies, i.e., antibodies capable ofbinding to the complex but do not substantially affect its biologicalactivities. A cannabinoid labeled with a detectable marker is contactedwith the immobilized TRPV2. Test compounds can be contacted with theimmobilized TRPV2 protein to allow binding to occur under standardbinding assay conditions. To identify compound binding to a TRPV2, onecan measure the detectable marker associated with TRPV2 or disassociatedfrom the TRPV2. A test compound that binds competitively with thelabeled cannabinoid to the TRPV2 will result in less binding of TRPV2 tothe cannabinoid, thus less labeling associated with TRPV2.

In one embodiment, the test compound can be further evaluated for itsability to increase or decrease the ion conductivity of a TRPV2 channel.Known to those skilled in the art are methods for measuring a TRPV2channel conductivity, for example, via the cellulardepolarization/hyperpolarization or an increase in intracellular calciumion levels. The level of intracellular calcium can be assessed using acalcium ion-sensitive fluorescent indicator, such as a calciumion-sensitive fluorescent dye. Suitable calcium ion-sensitivefluorescent dyes include, for example, quin-2 (see, e.g., Tsien et al.,J. Cell BioL, 94:325, 1982), fura-2 (see, e.g., Grynkiewicz et al., J.BioL Chem., 260:3440, 1985), fluo-3 (see, e.g., Kao et al., J. BioL-43Chem., 264:8179, 1989) and rhod-2 (see, e.g., Tsien et al., J. BioLChem., Abstract 89a, 1987). Suitable calcium ion-sensitive fluorescentdyes are commercially available from, for example, Invitrogen (MolecularProbes Products, Eugene, Oreg.). Cellular fluorescence can also bemonitored using a fluorometer or a flow cytometer having a fluorescencelamp and detector. FLIPR assay has been used routinely in measuring theion conductivity.

The TRPV2 cation channels function to transport not only divalentcations, for example, Ca²⁺, but also monovalent cations, for example,Na⁺ or K⁺. Therefore, assays for determining changes in the transport ofmonovalent cation can also be performed to measure the conductivity of aTRPV2 channel. Na⁺- and K⁺-sensitive dyes are known in the art andcommercially available from, for example, Invitrogen (Molecular ProbesProducts, Eugene, Oreg.).

The conductivity of a TRPV2 channel can also be measured byelectrophysiologic techniques such as patch clamp. Patch clamptechniques are routinely used for studying electrical activities incells, cell membranes, and isolated tissues. It involves forming anelectrically tight, high-resistance seal between a micropipette and theplasma membrane. The current flowing through individual ion channelswithin the plasma membrane can then be measured. Different variants onthe techniques allow different surfaces of the plasma membrane to beexposed to the bathing medium. The four most common variants includecell-attached, inside-out, outside-out and whole-cell patch clamp.

A patch-clamp method is commonly used with a voltage clamp that controlsthe voltage across the membrane and measures current flow. For example,in the case of whole-cell patch clamp, during the voltage clamp process,a microelectrode is inserted into a cell and current injected throughthe electrode so as to hold the cell membrane potential at somepredefined level. A patch-clamp method can also be used in thecurrent-clamp configuration, in which the current is controlled and themembrane potential is measured.

In another embodiment, the test compound can be further evaluated byadministering it to a live animal. This can be useful to establishefficacy, toxicity and other pharmacological parameters important forestablishing dosing regimens. For example, the compound can beadministered to a dog to examine various pharmacological aspects of thecompound in the dog. The dog testing can be particularly advantageousfor identifying and establishing dosing regimens in humans, becausedogs, particularly large breeds, are closer in weight to humans ascompared to rats or mice and therefore provide a more suitable animalmodel for estimating human dosing.

The compound can also be administered to animals to assess the abilityof the compound to alter nociceptive processes. Various animal models ofpain exist. For example, the rat spinal nerve ligation (SNL) model ofnerve injury is a model of neuropathic pain (Kim and Chung, Pain,50:355-363, 1992).

Other suitable animal models of pain can be utilized in connection withthe teachings herein. Commonly studied rodent models of neuropathic paininclude the chronic constriction injury (CCI) or the Bennett model;neuroma or axotomy model; and the partial sciatic transection or Seltzermodel (Shir et al., Neurosci. Lett., 115:62-67, 1990). Exemplaryneuropathic pain models include several traumatic nerve injurypreparations (Bennett et al, Pain 33: 87-107, 1988; Decosterd et al.,Pain 87: 149-58, 2000; Kim et al, Pain 50: 355-363, 1992; Shir et al.,Neurosci Lett 115: 62-7, 1990), neuroinflammation models (Chacur et al.,Pain 94: 231-44, 2001; Milligan et al., Brain Res 861: 105-16, 2000),diabetic neuropathy (Calcutt et al., Br J Pharmacol 122: 1478-82, 1997),virus-induced neuropathy (Fleetwood-Walker et al., J Gen Virol 80:2433-6, 1999), vincristine neuropathy (Aley et al., Neuroscience 73:259-65, 1996; Nozaki-Taguchi et al., Pain 93: 69-76, 2001), andpaclitaxel neuropathy (Cavaletti et al., Exp Neurol 133: 64-72, 1995),as well as acute nociceptive models and inflammatory models (Brennan, T.J. et al. Pain 64:493, 1996; D'Amour, F. E. and Smith, D. L. J Pharmacol72: 74-79, 1941; Eddy, N. B. et al. J Pharmacol Exp Ther 98:121, 1950;Haffner, F. Dtsch Med Wochenschr 55:731, 1929; Hargreaves, K. et al.Pain 32: 77-88, 1988; Hunskaar, S. et al. J Neurosci Meth 14:69, 1985;Randall, L. O. and Selitto, J. J. Arch. Int. Pharmacodyn 111: 409-419,1957; Siegmund, E. et al. Proc Soc Exp Bio Med 95:729, 1957).

The discovery that certain cannabinoids activate TRPV2 also provides newmethods for identifying additional compounds that increase thebiological activity of TRPV2. Such methods can be based on rational drugdesign. Structural analogs or mimetics of the cannabinoid can beproduced based on rational drug design with the aim of improving drugpotency, efficacy and stability, and reducing side effects. Methodsknown in the art for rational drug design can be used in the presentinvention. See, e.g., Hodgson et al., Bio/Technology, 9:19-21 (1991);U.S. Pat. Nos. 5,800,998 and 5,891,628, all of which are incorporatedherein by reference.

Molecular modeling programs can be used to determine whether a smallmolecule can fit into a functionally relevant portion, for example, anactive site, of the TRPV2 polypeptide. Basic information on molecularmodeling is provided in, for example, M. Schlecht, Molecular Modeling onthe PC, 1998, John Wiley & Sons; Gans et al., Fundamental Principals ofMolecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen (editor),Guidebook on Molecular Modeling in Drug Design, 1996, Academic Press;and W. B. Smith, Introduction to Theoretical Organic Chemistry andMolecular Modeling, 1996. U.S. patents that provide detailed informationon molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080,576;5,612,894; and 5,583,973.

Programs that can be useful for molecular modeling studies include, forexample, GRID (Goodford, P. J., “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules” J. Med. Chem., 28, pp. 849-857, 1985),available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M.Karplus, “Functionality Maps of Binding Sites: A Multiple CopySimultaneous Search Method.” Proteins: Structure, Function and Genetics,11, pp. 29-34, 1991), available from Molecular Simulations, Burlington,Mass.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Docking ofSubstrates to Proteins by Simulated Annealing” Proteins: Structure.Function, and Genetics, 8, pp. 195-202, 1990); available from ScrippsResearch Institute, La Jolla, Calif.; and DOCK (Kuntz, I. D. et al., “AGeometric Approach to Macromolecule-Ligand Interactions” J. Mol. Biol.,161, pp. 269-288, 1982), available from University of California, SanFrancisco, Calif.

In this respect, structural information on the TRPV2-cannabinoid complexis obtained. Preferably, atomic coordinates defining a three-dimensionalstructure of the complex can be obtained. For example, the interactingTRPV2-cannabinoid complex can be studied using various biophysicaltechniques including, e.g., X-ray crystallography, NMR, computermodeling, mass spectrometry, and the like. Likewise, structuralinformation can also be obtained from protein complexes formed byinteracting proteins and a compound that initiates or stabilizes theinteraction of the proteins. Methods for obtaining such atomiccoordinates by X-ray crystallography, NMR, and the like are known in theart and the application thereof to the target protein or protein complexof the present invention should be apparent to skilled persons in theart of structural biology. See Smyth and Martin, Mol. Pathol., 53:8-14(2000); Oakley and Wilce, Clin. Exp. Pharmacol. Physiol., 27(3):145-151(2000); Ferentz and Wagner, Q. Rev. Biophys., 33:29-65 (2000); Hicks,Curr. Med. Chem., 8(6):627-650 (2001); and Roberts, Curr. Opin.Biotechnol., 10:42-47 (1999).

The domains, residues or moieties of a cannabinoid critical toTRPV2-cannabinoid interaction constitute the active region of thecannabinoid known as its “pharmacophore.” Once the pharmacophore hasbeen elucidated, a structural model can be established by a modelingprocess that may incorporate data from NMR analysis, X-ray diffractiondata, alanine scanning, spectroscopic techniques and the like. Varioustechniques including computational analysis (e.g., molecular modelingand simulated annealing), similarity mapping and the like can all beused in this modeling process. See e.g., Perry et al., in OSAR:Quantitative Structure-Activity Relationships in Drug Design, pp.189-193, Alan R. Liss, Inc., 1989; Rotivinen et al., Acta PharmaceuticalFennica, 97:159-166 (1988); Lewis et al., Proc. R. Soc. Lond.,236:125-140 (1989); McKinaly et al., Annu. Rev. Pharmacol. Toxiciol.,29:111-122 (1989). Commercially available molecular modeling systemsfrom Polygen Corporation, Waltham, Mass., include the CHARMm program,which performs energy minimization and molecular dynamics functions, andQUANTA program, which performs construction, graphic modeling andanalysis of molecular structure. Such programs allow interactiveconstruction, modification, and visualization of molecules. Othercomputer modeling programs are also available from BioDesign, Inc.(Pasadena, Calif.), Hypercube, Inc. (Cambridge, Ontario), and Allelix,Inc. (Mississauga, Ontario, Canada).

A template can be formed based on the established model. Variouscompounds can then be designed by linking various chemical groups ormoieties to the template. Various moieties of the template can also bereplaced. In addition, in the case of a peptide lead compound, thepeptide or mimetics thereof can be cyclized, e.g., by linking theN-terminus and C-terminus together, to increase its stability. Theserationally designed compounds are further tested. In this manner,pharmacologically acceptable and stable compounds with improvedpotency/efficacy and reduced side effects can be developed. Thecompounds identified in accordance with the present invention can beincorporated into a pharmaceutical formulation suitable foradministration to an individual.

In addition, the structural models or atomic coordinates defining athree-dimensional structure of the target protein or protein complex canalso be used in virtual screen to select compounds capable of activatingTRPV2. Various methods of computer-based virtual screen using atomiccoordinates are generally known in the art. For example, U.S. Pat. No.5,798,247 (which is incorporated herein by reference) discloses a methodof identifying a compound (specifically, an interleukin convertingenzyme inhibitor) by determining binding interactions between an organiccompound and binding sites of a binding cavity within the targetprotein. The binding sites are defined by atomic coordinates.

The compounds designed or selected based on rational drug design orvirtual screen can be tested for their ability to modulate (interferewith or strengthen) the interaction between the interacting partnerswithin the protein complexes of the present invention. In addition, thecompounds can further be tested in TRPV2 channel conductivity assays oranimal models as described supra.

Following the selection of desirable compounds according to the methodsdisclosed above, the methods of the present invention further providefor the manufacture of the selected compounds. The compounds can bemanufactured for further experimental studies, or for therapeutic use.The compounds identified in the screening methods of the presentinvention can be made into therapeutically or prophylactically effectivedrugs for preventing or ameliorating diseases, disorders or symptomscaused by or associated with TRPV2, such as pain, or inflammation, etc.

Example 1 Expression of Rat and Human TRPV2 in HEK293 Cells

A cDNA fragment encoding the full-length rat TRPV2 was subcloned intopCI-neo (Promega, Madison, Wis.) mammalian expression vector. Theexpression construct was then transfected into HEK293 cells with FuGene6transfection reagent (Roche, Indianapolis, Ind.) according to thevendor's protocol. Stable cell lines were selected by growth in thepresence of 400 μg/ml G418. Single G418 resistant clones were isolatedand purified. Stable expression of rat TRPV2 in these cells wasconfirmed by Western blot analysis with an anti-rat TRPV2 specificantibody (Chemicon, Temecula, Calif.), Ca²⁺ imaging assay (FLIPR) andwhole cell patch clamp analyses.

A cDNA fragment encoding the full-length human TRPV2 was subcloned intopCI-neo or pcDNA3 mammalian expression vectors. The expressionconstructs were then transfected into HEK293 cells with FuGene6transfection reagent (Roche, Indianapolis, Ind.) according to vendor'sprotocol. TRPV2-expressing HEK293 cells were cultured in DMEMsupplemented with 10% fetal bovine serum, 100 units/ml penicillin, and100 μg/ml streptomycin for 48-72 hr and either evaluated for transientexpression and/or activity, or dosed with 400 μg/ml G418 to select forstably-transfected TRPV2-expressing cell clones. Cells were maintainedat 37° C. and in 5% CO₂.

Example 2 TRPV2 is activated by Δ⁹-tetrahydrocannabinol (Δ⁹-THC)

To search for pharmacological activators of TRPV2, Δ⁹-THC, a majorpsychoactive constituent of marijuana derived from Cannabis, was tested.The rat TRPV2-expressing HEK293 cells were seeded in a 384-well plate ata concentration of 5×10³ cells/well and incubated overnight at 37° C.The following day, the cells were loaded with buffer and calcium dye 3(Molecular Devices, Sunnyvale, Calif.) in a final volume of 50 μl andincubated for 30 minutes at 37° C./5% CO₂ followed by 30 additionalminutes at room temperature. The fluorescence intensity was measured bya fluorescent plate reader (FLIPR) before, during and after the additionof test compounds.

As shown in FIG. 4A, addition of 100 μM Δ⁹-THC solid line but not buffer(dotted line), caused a robust elevation of intracellular Ca²⁺ in ratTRPV2-expressing HEK293 cells. In contrast, no significant intracellularCa²⁺ elevation was observed in untransfected HEK293 cells at the sameconcentration of Δ⁹-THC (dashed line), suggesting that the elevation ofintracellular Ca²⁺ was mediated by rat TRPV2. Activation of rat TRPV2 byΔ⁹-THC was dose-dependent with an EC₅₀ value of 15.7 uM and Hill slopeof 1.04 (FIG. 4B).

To further confirm the Δ⁹-THC effect on TRPV2, whole-cell patch clampstudies were performed. The extracellular solution contained (in mM):NaCl, 132; CaCl, 1.8; KCl, 5.4; MgCl₂, 0.8; HEPES, 10; glucose, 10;pH=7.4. The intracellular solution used to fill recording pipettescontained (in mM): CsCl, 145; EGTA, 5; HEPES, 10; glucose, 5; pH=7.4.Recordings were performed using the conventional whole-cell patch clamptechnique, 2-3 days after transient transfection of human TRPV2 intoHEK293 cell or 1-2 days after plating HEK293 cells stably expressing ratTRPV2 onto glass coverslips. Currents were amplified by a patch clampamplifier and filtered at 2 kHz (Axopatch 200B), sampled at 10 kHz usingDigidata 1322A and acquired and analyzed with pClamp 9.0 (allinstruments from Molecular Devices, CA). A 600 ms voltage ramp was givenonce every five seconds from −100 mV to +60 mV. The holding potentialbetween voltage ramps was −100 mV. Extracellular solutions were appliedto the cell at 0.5 ml/min via a gravity-fed perfusion system. Allexperiments were performed at 22° C.

As shown in FIG. 5B, upon application of 100 μM Δ⁹-THC, there was asignificant increase of the whole-cell current amplitude (gray solidline) in HEK293 cells expressing rTRPV2 compared to control (blackdotted line) at both hyperpolarized and depolarized membrane potentials.However, this effect was more pronounced at depolarized potentials. Thesame concentration of Δ⁹-THC elicited no current above control level inuntransfected HEK293 cells (data not shown). The Δ⁹-THC-activatedcurrent had a reversal potential near 0 mV, indicating the relativelyunselective (at least for the cations used in these experiments) natureof the channel. Similar effects induced by Δ⁹-THC were also observed inhuman TRPV2 (FIG. 5A). Furthermore, the Δ⁹-THC-activated currents weresignificantly inhibited by 10 μM ruthenium red (RR), a non-selective TRPchannel inhibitor (black line) in both rat and human TRPV2. Takentogether, these results indicate that Δ⁹-THC activates currents mediatedby TRPV2 in these cells.

Example 3 TRPV2 is Activated by Other Cannabinoids

To further explore activation of TRPV2 by cannabinoids, severaldifferent classes of cannabinoids were tested in a FLIPR calciummobilization assay using 100 μM compound concentrations (except foranandamide which was at 120 μM) as evaluated using rat TRPV2-expressingHEK293 cells. All data was normalized to that observed for 100 μMΔ⁹-THC. The tested compounds included: the non-psychoactive constituentsof marijuana (cannabidiol and cannabinol); synthetic analogs of THC(nabilone, CP 55,940, HU210, HU211, HU-308, HU331, 11-hydroxy-Δ9-THC,and O-1821); several endocannabinoids (anandamide,2-arachidonoyl-glycerol (2-AG) and their analog palmitoylethanolamide(PEA)); a cannabinoid transport blocker (AM404); other syntheticcannabinoid receptor agonists (WIN55, 212-2, WIN55, 212-3, JWH015,JWH133, O-1918 and CAY10429); and the non-selective agonist, 2-APB Theability of these compounds to activate rat TRPV2 is shown in Table 1.The data suggest that TRPV2 could be activated by more than one class ofcannabinoids.

TABLE 1 Activation of rat TRPV2 by Cannabinoids Compound CompoundStructure % Response EC₅₀ Δ⁹-THC

100 15.5 uM Cannabinol

68 77.7 uM Nabilone

59 CP55,940

43 HU-210

39 WIN-55,212-2

−2 JWH015

14 Anandamide

0 2-AG

30 PEA

5 AM404

6 Cannabidiol

163 3.7 uM 11-hydroxy-Δ⁹-THC

58 O-1821

95 ~20 uM O-1918

6 CAY10429

0 WIN 55,212-3

7 HU-211

31 HU-308

10 HU-331

47 ~14 uM JWH-133

5 2-APB

102 8.0 uM

Example 4 Δ⁹-THC Selectively Activates TRPV2

A selected number of cannabinoids were also tested against HEK293 cellsexpressing human TRPV1 and canine TRPM8 in the FLIRP assay. Assummarized in Table 2, Δ⁹-THC (240 μM) and cannabinol (600 μM) showed nosignificant activation of TRPV1, whereas anandamide, 2-AG and AM404activated TRPV1, consistent with previous reports. None of thesecannabinoids showed agonist effect against TRPM8.

TABLE 2 Activation of TRP channels by cannabinoids TRPV1 TRPV2 TRPM8Δ⁹-THC (240 μM) − + − Cannabinol (600 μM) − + − Anandamide (120 μM) + −− 2-AG (120 μM) + + − AM404 (120 μM) + − −

Example 5 Deletion Mutants of TRPV2 were Activated by Cannabinoids

TRPV2 deletion mutants were constructed and tested for activation byΔ⁹-THC, 2-APB and high temperature stimulation. Methods of this Examplecan be used to construct and test any type of TRPV2 deletion mutants,including, but not limited to, mutants having one or more amino acidresidues deleted at the N-terminal of the TRPV2, at the C-terminal ofthe TRPV2, and/or at any other location of the TRPV2.

DNA molecules encoding the deletion mutants were amplified by PCR usingthe rat TRPV2 cDNA as the template. For amino-terminal deletions, aseries N-terminal forward primers encoding an initiating methionine inframe with sequences matching adjacent regions of the desired start atresidues G21 (mutant N20, SEQ ID NO:11), P33 (N32, SEQ ID NO:12), A66(N65, SEQ ID NO:13) and V84 (N83, SEQ ID NO:14) paired with a C-terminalreverse primer were used for PCR amplification. While forcarboxyl-terminal deletions, a forward N-terminal primer paired with aseries C-terminal reverse primers ending at residues R706 (C51, SEQ IDNO:7), P729 (C32, SEQ ID NO:8), P738 (C23, SEQ ID NO:9) and E750 (C11,SEQ ID NO: 10) with an in-frame stop codon at the terminal end of theopen reading frame were used for amplification. After PCR amplificationand purification, the DNA molecules encoding the deletion mutants weresubcloned into the pCI-neo mammalian expression vector and theconstructs were confirmed by DNA sequencing. The DNA molecules thatencoded for the various TRPV2 deletion mutants comprised the nucleotidesequences of: SEQ ID NO:18 (N20), SEQ ID NO:19 (N32), SEQ ID NO:20(N65), SEQ ID NO:21 (N83), SEQ ID NO:22 (C51), SEQ ID NO:23 (C32), SEQID NO:24 (C23), and SEQ ID NO:25 (C11).

The deletion mutant constructs were then transfected into HEK293 cellsusing Fugene 6 reagent (Roche) as per the manufacturer's instructions.At 24 hours post-transfection, the cells were harvested and replated infresh DMEM medium supplemented with 10% fetal bovine serum, 100 units/mlpenicillin, 100 μg/ml streptomycin and. The cells were distributed ontopoly-D-Lysine coated 96- or 384-well plates at a density ofapproximately 40,000 and 10,000 cells per well, respectively. Atapproximately 48 hours post-transfection, the medium was removed fromthe assay plate and replaced with Calcium 3 Dye Buffer (MolecularDevices) using the protocol available from the manufacturer. Calciummobilization was triggered using Δ⁹-THC or 2-APB or elevated temperaturebuffer and measured using either FLIPR or FLEX STATION instruments.

It was found that deletion mutants of rat TRPV2 lacking the N-terminal20, 33, 66, or lacking the C-terminal 11, 23, or 32 amino acid residueswere still active in their responses to Δ⁹-THC (FIG. 6), 2-APB and anelevated temperature of about 53° C. (data not shown).

Example 6 Activation of TRPV2 Chimera

The domain-swapping chimeras between rat and human TRPV2s were also madeand tested for activation by Δ⁹-THC, 2-APB and high temperaturestimulation. Methods of this Example can be used to construct and testany type of TRPV2 chimeras, including, but not limited to, thedomain-swapping chimeras between TRPV2s from different animals, and thechimeras between TRPV2 and other TRPV channels such as TRPV1 and TRPV3.

Based on the predicted topology and primary sequence features of TRPV2,TRPV2 are divided into 3 major domains: 1) the amino-terminalintracellular domain; 2) the transmembrane domain; and 3) thecarboxyl-terminal intracellular domain. For a chimera, each domain canbe of rat (R) or human (H) origin. Three rat and human TRPV2 chimerawere constructed and tested herein. RRH (SEQ ID NO:15) was a chimeracomprising rat 1-392 aa, rat 393-646 aa, and human 647-764 aa. RHR (SEQID NO:16) was a chimera comprising rat 1-392, human 391-646, and rat647-761. HRR (SEQ ID NO:17) was a chimera comprising human 1-390, rat393-646, and rat 647-761.

DNA molecules encoding the three rat and human TRPV2 chimeras wereobtained by fusion PCR. First, DNA molecules encoding the desired TRPV2domains were amplified by PCR using the rat or human TRPV2 cDNA as thetemplate with synthetic primer DNA containing in-frame sequence thatoverlapped with the other species domain to be linked. After PCRamplification and purification, DNA molecules encoding the desired TRPV2domains from human and rat were combined and used as templates forfusion PCR with primer DNA matching the 5′ and 3′ end sequences of thecoding sequence for the full length TRPV2 chimera. The DNA moleculesthat encoded the various TRPV2 chimeras comprised the nucleotidesequences of: SEQ ID NO: 26 (RRH), SEQ ID NO:27 (RHR), and SEQ ID NO:28(HRR).

The DNA molecules encoding the TRPV2 chimera were then subcloned intothe mammalian expression vector, pCI-neo and the resulting constructsconfirmed by DNA sequencing. The chimeras were transiently expressed inHEK293 cells and their responses to a variety of stimulators were alsotested as described in Example 5.

Chimera RHR was fully responsive to the addition of Δ⁹-THC (FIG. 7A),).Although the HEK cells expressing chimera RRH or HRR separately werepoorly, or not active, respectively, the cells co-expressing bothchimeras (RRH+HHR) were fully responsive to Δ⁹-THC (FIG. 7B. Similarresponses by the above-listed chimeras to 2-APB stimulation wereobserved (data not shown). The gain of function study by coexpression oftwo inactive mutants suggests that a functional TRPV2 channel is acomplex with multiple subunits and some of the critical functionaldomains act in trans rather than in cis.

1-18. (canceled)
 19. A method for identifying a compound that increasesthe biological activity of TRPV2, comprising the steps of a. obtainingatomic coordinates defining a three-dimensional structure of a complexcomprising a TRPV2 interacting with a cannabinoid that is capable ofactivating the TRPV2; b. elucidating a structural relationship betweenthe TRPV2 and the interacting cannabinoid; c. designing a structuralanalog of the cannabinoid based on the structural relationship; d.synthesizing the structural analog; e. determining the extent to whichthe structural analog alters the biological activity of the TRPV2,thereby identifying the compound that increases the biological activityof TRPV2.
 20. The method of claim 19, wherein the biological activity ofthe TRPV2 is determined as calcium-influx into a cell expressing theTRPV2.
 21. The method of claim 19, wherein the biological activity ofthe TRPV2 is measured by a method of patch clamp.
 22. The method ofclaim 19, wherein the biological activity of the TRPV2 is measured by aCA mobilization assay.
 23. The method of claim 19, wherein thebiological activity of the TRPV2 is determined as its binding affinityto the cannabinoid that is capable of activating the TRPV2 activity. 24.A method of increasing the biological activity of a TRPV2, comprisingthe step of contacting the TRPV2 with a cannabinoid that is capable ofactivating the TRPV2 activity.
 25. The method of claim 24, wherein theTRPV2 is associated with an isolated membrane.
 26. The method of claim24, wherein the TRPV2 is present in a cell.
 27. The method of claim 26,wherein the cell is a neuron.
 28. The method of claim 24, wherein thecannabinoid is selected from the group consisting ofΔ⁹-tetrahydrocannabinol, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol,cannabidiol, O-1821, nabilone, CP55940, 2-AG, and HU210, HU211, HU308,and HU331.
 29. A method for stimulating noxious thermo-sensation in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising an effective amount of cannabinoid that iscapable of activating the TRPV2 activity, thereby stimulating thenoxious thermo-sensation in the subject.
 30. The method of claim 29,wherein the subject is a human.
 31. The method of claim 29, wherein thecannabinoid is selected from the group consisting ofΔ⁹-tetrahydrocannabinol, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol,cannabidiol, O-1821, nabilone, CP55940, 2-AG, and HU210, HU211, HU308,and HU331.
 32. An isolated polypeptide consisting essentially of anamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, or SEQ ID NO:17.
 33. An isolated nucleic acidmolecule that encodes a polypeptide consisting essentially of an aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or SEQ ID NO:17.
 34. The isolated nucleic acid molecule ofclaim 33 consisting essentially of a nucleotide sequence of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO:27, or SEQID NO:28.
 35. An expression vector comprising nucleotide sequence thatencodes a polypeptide consisting essentially of an amino acid sequenceof SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, orSEQ ID NO:17.
 36. A recombinant cell comprising an expression vector ofclaim
 35. 37. A method of producing a polypeptide consisting essentiallyof an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, comprising the step ofgrowing a cell of claim 36 under a condition whereby the polypeptide isproduced by the cell.